Amperometric biosensors are capable of quantifying trace amounts of biological analytes such as glucose, urea, cholesterol, and the like in biological fluids. Analyte may be electroxidized directly at the electrode, or an enzyme may be immobilized on an electrode such that the reaction product of the enzyme with its substrate is detected by an electrical change, e.g., change in current flow, at the electrode. The current generated at the electrode is a function of the quantity of analyte in a sample.
Historically, electrical communication between enzyme and electrode has been achieved through the use of diffusing mediators. These include small diffusing redox shuttles such as ferrocenes, quinones, ruthenium amines, and the like. Enzyme electrodes using such mediators generally require that the enzyme and mediator shuttle be confined to the proximity of the electrode surface. The small shuttle molecules commonly employed can, however, readily diffuse through membranes that contain the enzyme. These membranes must allow passage of the enzyme's substrate, e.g., glucose and product e.g., gluconate or gluconolactone. Diffusion of the mediators away from the electrode surface results in reduced sensitivity of the electrode.
The inventors have previously reported the production of novel amperometric biosensors having a three-dimensional redox polymer network to which a redox enzyme is chemically bound. The three-dimensional redox molecular structure provides electrical contact between the surface of the electrode and the redox enzyme. Such electrodes are described in co-pending application U.S. Ser. No. 389,226 which is hereby incorporated by reference as if fully set forth and in Gregg and Heller, J. Phys. Chem., 95:5970-5980 (1990) and Heller, Accounts of Chem. Res., 23:128 (1990).
The three-dimensional redox network of these electrodes is preferably formed of a redox enzyme, a cross-linking agent, and a cross-linkable compound capable of reacting with the cross-linking agent and the redox enzyme. Either the cross-linkable compound or the cross-linking agent, or both, have multiple redox centers. When the compounds of each embodiment are mixed together under appropriate conditions, a chemical reaction takes place resulting in the formation of a cross-linked, three dimensional, redox polymer with the redox enzymes chemically bound within the cross-linked redox polymer network.
Materials known to produce the three-dimensional redox polymer network and effect the directly wired enzyme electrodes described above are limited. It would be desirable to have provided a variety of materials from which such electrodes could be produced and/or improved.
It has recently been found that the sensitivity of the three-dimensional redox polymer network electrodes described above in biological fluids such as serum or blood is decreased by a factor of 10-100 within 10 seconds as compared with sensitivity in a buffer solution. After loss of sensitivity in such a biological sample, the original sensitivity is recovered if the electrode is rinsed and retested in buffer.
It would be highly desirable to prevent this reversible loss of sensitivity of the three-dimensional redox polymer network enzyme electrodes to obtain an improved enzyme electrodes having high sensitivity.